The present invention is related to a diagnostic system and to a method for determining the presence of a genotoxic compound in a sample.
The International Patent Application PCT/EP96/01745 describes a recombinant nucleic acid sequence, a host micro-organism comprising said nucleic acid sequence and the use of said host micro-organism for detecting the presence of genotoxic compounds in a sample. Said bacterial genotoxicity test is based on bioluminescence and allows an easy, very rapid and low cost detection of genotoxic compounds. The test was shown to be at least as sensitive as the Ames test and SOS-chromotest and to allow genotoxicity kinetics measurements as well as a simultaneous evaluation of the toxicity of the test compound or -material (van der Lelie et al., 1997). This new test, referred to as the VITOTOX(copyright) test was therefore considered to be a valuable short-term genotoxicity and toxicity test for many different purposes.
The test is based on bacteria that contain the lux operon of Vibrio fischeri under transcriptional control of the recN gene, that is part of the SOS-system. After incubation of the bacteria in the presence of a genotoxic compound, the recN promoter is derepressed, resulting in expression of the lux operon. This expression results in light production in function of genotoxicity. Originally, the test was performed with different modified Escherichia coli and Salmonella typhimurium strains. Salmonella typhimurium strains (TA98, TA100 and TA104) were further used, as the bacteria are well known for mutagenicity testing and because the same bacteria could also be used for a classical Ames test, should this be required. The construct using a recN promoter up mutation (recN 2-4) gave the best results in all strains. Furthermore, as all Salmonella strains gave very comparable results, it has been proposed to further use only the TA104 constructs [called TA104 (recN2-4)] as it was shown to be sometimes a little more sensitive than the other hybrid strains (van der Lelie et al., 1997).
However, some compounds act directly on the light production (e.g. aldehydes, organic solvents) or enhance the metabolism of the bacteria creating false-positive results.
The present invention aims to provide a new diagnostic system and method for the detection of environmental insults such as the presence of a genotoxic compound into a sample, which do not present the drawbacks of the state of the art.
A main aim of the present invention is to provide a new diagnostic system and method which will exclude false-positive and false-negative results.
A further aim of the present invention is to provide such a diagnostic system and method which could be used for the screening of genotoxic compounds obtained in the chemical, cosmetical or pharmaceutical industry field, as a prevalidation study of intermediate or active chemical, cosmetical and/or pharmaceutical compounds.
A last aim of the present invention is to provide such a diagnostic system and method which allow an automatic screening upon very small volume of sample.
The present invention is related to a diagnostic system made of
a transformed micro-organism capable of an increased reporter activity upon exposure to an environmental insult, preferably exposure to a genotoxic compound, said micro-organism having a stress inducible promoter sequence, preferably a promoter sequence which is inducible by a genotoxic compound, said promoter sequence being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter molecule resulting in a signal that can be assayed, and of
a transformed micro-organism having a constitutive and non-stress inducible promoter sequence, preferably a constitutive promoter sequence which is not inducible by said genotoxic compound, said promoter sequence being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter molecule resulting in a signal that can be assayed.
Preferably, both transformed micro-organisms are bioluminescent micro-organisms and the signal can be assayed as light production. Other possibilities are the peroxydase, alkaline phosphatase, xcex2-gal and gus genetic sequence, where the signal will be assayed as a colorimetric modification or a chemiluminescent light production by using a colorimetry analyser or a photomultiplier device.
Advantageously, the diagnostic system according to the invention is made of two transformed bioluminescent micro-organisms, the first bioluminescent micro-organism is xe2x80x9cactivatedxe2x80x9d in the presence of a genotoxic compound and results in a signal that can be assayed as light production, while the light production of the second bioluminescent micro-organism is not influenced by the presence of a genotoxic compound in the sample.
Nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production has been already described in the state of the art. Preferably, the transformed bioluminescent micro-organisms according to the invention comprise luciferase A and B genes, also identified as expressive lux genes complex, comprising the luxA and luxB genes or a translational luxAB fusion gene. The transformed bioluminescent micro-organisms may also comprise the luciferase C, D and E genes, required for production of limiting fatty acid substrate that is used in recycling.
According to a preferred embodiment of the present invention, the diagnostic system comprises a transformed bioluminescent micro-organism having a constitutive and non stress inducible promoter sequence with a Sigma 70 consensus sequence (TTGACA(xe2x88x9235) . . . 17/18 bp . . . TATAAT(xe2x88x9210)) and whose transcription is not regulated positively or negatively at the promoter level. Said promoter consensus is described in Hoopes B C and McClure W R (1987): Strategies in Regulation of Transcriptional Initiation; in xe2x80x9cEscherichia Coli and Salmonella typhimurium, Cellular and Molecular Biology, FC Neidhart, J L Ingraham, K B Low, B Maganasik, M Schaechter, H E Umbaeger (eds.), American Society for Microbiology, Washington D.C., pp 1231-1240.
According to a preferred embodiment of the present invention, the transformed bioluminescent micro-organisms are selected from the group consisting of E. coli or Salmonella typhimurium, and are advantageously suitable Ames test micro-organism(s), preferably selected from the group consisting of TA98, TA100, TA102, TA104, TA1535, TA1538, TA7001 to TA7006, and TA7041 to TA7046, and/or having the micro-organism deposit number LMG P-18318. The micro-organism with deposit number LMG P-18318 will be identified hereafter as the xe2x80x9cpr1xe2x80x9d strain.
Advantageously, the stress inducible promoter sequence in the transformed bioluminescent micro-organism of the diagnostic system according to the invention is selected from the group consisting of groEL, dnaK, grpE, phoA, glnA, lon, lysU, rpoD, clpB, clpP, uspA, katG, uvrA, frda, micF, fabA, lac, his, sodA, sodB, soi-28, recA, xthA, narG, recF, recj, recN, recO, recQ, ruv, uvrD, ars, cad, mer, pco, and sfiA.
According to a preferred embodiment of the present invention, the stress inducible promoter sequence is recN, advantageously recN2-4. Said micro-organism will be identified hereafter as the xe2x80x9crecN2-4xe2x80x9d strain
In a preferred embodiment, the diagnostic system according to the invention comprises a transformed bioluminescent micro-organism having a stress inducible promoter sequence being a SOS regulator promoter sequence, having preferably an induction ration higher than 40, and comprising advantageously a mutation improving the promoter strength or regulation, said mutation not destroying the SOS regulation.
A specific example of mutated recN promoter sequence is described in the International Patent Application PCT/EP96/01745, incorporated hereafter by reference.
Said stress inducible promoter sequence comprises also a promoter up-mutation, preferably a promoter up-mutation in the xe2x88x9235 region of said promoter, described in the International Patent Application PCT/EP96/01745, incorporated hereafter by reference.
The present invention is also related to a diagnostic kit comprising the elements of the diagnostic system according to the invention and possibly the necessary additional reactants, diluants and/or solid supports for said diagnostic such as a buffer solution, a solution comprising a specific marker such as the X-gal (5-bromo-4-chloro-3-indoyl-xcex2-galactoside) for a diagnostic based upon the use of a xcex2-gal genetic sequence, the luminol for a diagnostic based upon the use of a peroxydase genetic sequence, etc . . .
The present invention is also related to a general method for the diagnostic of an environmental stress or insult, preferably for determining the presence of a genotoxic compound in a sample. Said method comprises the steps of exposing the diagnostic system according to the invention to said environmental insult (preferably comprising the steps of exposing the diagnostic system to the genotoxic compound present in the sample) and measuring a signal, preferably a change in luminescence of said diagnostic system.
The present invention is also related to a method for determining the kinetics of genotoxicity of a compound into a sample, based upon the above-identified method, wherein the measuring of luminescence of both inducible and constitutive transformed micro-organisms occurs at multiple points in time, preferably continuously, and in addition carrying out the step of determining the signal-to-noise (S/N) ratio for the transformed micro-organisms at said point and time, dividing the S/N ratio of the inducible micro-organism by that of the constitutive micro-organisms and plotting these data, said plotting representing the corrected kinetics of genotoxicity of the genotoxic compound in the sample.
Advantageously, the diagnostic system and method according to the invention can be combined with the Ames and/or SOS chromotest(s) and method(s), as described hereafter.
The present invention is also related to an analysis method of an environmental insult, comprising the steps of:
performing the diagnostic of said environmental insult as above described,
calculating the signal to noise ratio of both the transformed micro-organisms,
classifying said environmental insult as toxic if at least one of the calculated signal to noise ratios is lower than 0.8,
classifying said environmental insult as having no effect if the signal to noise ratio of the micro-organism comprising the stress-inducible promoter sequence lies between 0.8 and 1.2, and
classifying said environmental insult as inducing and genotoxic if the signal to noise ratio of the micro-organism comprising the stress-inducible promoter sequence is higher than 1.2 and is at least 50% higher than the signal to noise ratio of the micro-organism comprising the constitutive promoter sequence.
The present invention is also related to an installation for determining if a compound in a sample is genotoxic and/or toxic, comprising a diagnostic system as above described induced by said sample, a detection system comprising a detection apparatus able to assay the signal from said diagnostic system, said detection system being connected to a computer programmed to carry out the analysis method as above described.
The present invention will be described in detailed in the non-limiting following examples, in reference to the following figures.